Effective and enduring therapies for most types of cancers remain problematic; in these cases, the development of chemoprevention strategies and tools to prevent or slow the initiation and development of malignancies promises to have far-reaching impacts on the field of oncology. One of the most effective and best documented chemopreventive strategies involves the use of tamoxifen to reduce breast cancer mortality and morbidity. Surprisingly, however, little is known about the mechanism by which a limited-term course of tamoxifen treatment has long-term protective effects. Using a mouse model of breast cancer, we have discovered a tamoxifen-induced signature gene expression pattern associated with a life-time protection from tumor development. Importantly, this pattern included down-regulation of a number of genes involved in cell proliferation and differentiation, and reduced epithelial cellularity was evident in the mammary glands of chemoprotected mice. These findings lead us to hypothesize that a central mechanism of tamoxifen chemoprevention involves influences on the developmental events that give rise to mature mammary epithelial cells. This hypothesis becomes even more compelling and exciting in light of emerging evidence pointing to the importance of mammary stem and progenitor cells in breast cancer. The objective of this application is to determine more accurately the nature of the perturbations in normal epithelial differentiation patterns induced by tamoxifen, and to identify which cells in the developmental hierarchy (stem, progenitor, and mature) are the major tamoxifen targets. In addition, signature gene expression patterns will be compared among these different mammary cell types to identify specific molecular pathways involved in tamoxifen chemoprevention. The specific aims of this proposal include: Aim 1. Determine the effects of short-term tamoxifen chemoprevention on the proportions of mammary nonepithelial cells as well as epithelial stem, progenitor and mature luminal and myoepithelial cells. Aim 2. Compare the relationship between signature gene expression patterns in the stromal and stem, progenitor and differentiated cell fractions in cancer prone mice that have received tamoxifen chemopreventive vs. placebo regimens. Aim 3. Identify the human breast cells that express one or multiple signature genes. Aim 4. Determine whether the mechanisms of tamoxifen chemoprevention involve: a) direct or indirect effects on stem and progenitor cell self-renewal and proliferation; b) the process of epithelial differentiation, or c) survival of mature epithelial cells. The overall importance and translatability of these studies will be addressed by comparing three different mouse models of breast cancer and through the use of stem, progenitor, and mature epithelial cells from human breast surgical samples. Identification of the cells and molecular pathways that are modulated by tamoxifen will reveal novel targets and strategies for the development of the next generation of chemical and dietary preventives, as well as furnish useful biomarkers of risk or preventive efficacy.